Hypercholesterolemia is a major risk factor for the development of coronary artery disease (CAD) and cerebral vascular disease (CVD). Increased plasma levels of low-density lipoprotein (LDL) leads to deposition of excess cholesterol in arteries, initiating atherosclerosis. While homeostatic mechanisms that regulate LDL uptake and de novo synthesis of cholesterol are well characterized, the cellular mechanisms that regulate trafficking of LDL cholesterol after internalization are not well understood. Evidence is emerging that the NPC1 and HE1 proteins participate in a common pathway for the efficient trafficking of internalized membrane cholesterol to the plasma membrane (PM) and to the ER. We hypothesize that NPC1 promotes formation of vesicles and/or tubules through the budding of the limiting endosomal membrane. NPC1-containing vesicles then traffic to the PM or ER to deliver their sterol cargo. The purpose of this project is to identify the molecular machinery in the sterol trafficking pathway and to test our hypothesis regarding this role of NPC1 in the sorting and distribution of internalized membrane cholesterol. This will be achieved by the following specific aims: (1) Use of a functional mammalian genetic screen to isolate Chinese hamster ovary (CHO) mutants with impaired intracellular trafficking of cholesterol, (2) Characterization and identification of the cholesterol trafficking defects in the mutant CHO cell lines, and (3) Compositional and functional analysis of proteins in the NPC1-containing late endosomal compartment, and examination of whether NPC1 membrane vesciulation is dependent on sterol concentrations and/or an intact sterol-sensing domain in NPC1. The studies outlined in this proposal will further our understanding of the critical role of NPC1 in cholesterol homeostasis. Furthermore, study of the function of gene products identified by our genetic screens and by compositional analysis of the NPC1-containing endosomal compartment may identify novel targets within the sterol transport pathway for pharmacologic therapy of CAD and CVD.